This invention relates to detectors for use in chromatographs.
In analytical procedures where a spectrophotometer is employed in a detection system for liquid chromatographs, a method has been proposed to employ a spectroscope to measure light of a predetermined wavelength having an absorption peak with respect to sample components. In this method where absorbancy of light at a single wavelength alone is measured, the signal-to-noise (S/N) ratio is low, and in order to enhance the detector sensitivity it is required to intensify the light source and enlarge the spectral slit width. However, intensity of the light source is limited by available light sources. Another difficulty is that enlarging the slit width will cause the relationship of absorbancy to sample concentration to deviate from a straight line, so that quantitative accuracy will suffer. A primary object of this invention is to avoid the problems of light source intensity and large slit width.
In conventional recording of chromatograph detector outputs, one is not readily certain whether a recorded peak is due to a single component completely eluted or due to plural individual peaks separated incompletely. This uncertainty cannot be avoided by using a single detecting signal for providing a chromatogram.
One way to resolve this problem is to use plural detecting means to record plural chromatograms. Even with this method, however, it is still difficult to correctly interpret the resulting chromatogram. Observed peaks cannot be judged at a glance as to whether they represent incompletely separated components.
In techniques using plural detecting means, it is noted that the overall sensitivity depends upon the respective absorption of different components which inherently differ from each other. Accordingly, with reference to an observed peak on a chromatogram obtained from plural outputs of plural detecting means, a ratio among the respective detector outputs at an identical time which results in a constant value during the time period of the peak from the peak rise to its fall is due to a single component. In contrast, if the peak is due to plural component peaks superimposed, the ratio will vary with time depending upon their progressive rate of separation.
Another object of this invention is to provide a technique to provide that observed peaks can be judged at a glance as to whether they represent incompletely separated components. With the technique of the invention, at selected times a ratio among the respective detection outputs is obtained and a peak is judged to be related to a single component if a rectangular curve is traced; or the same peak is judged to be resulting from plural incompletely separated components if a curved curve is traced.
Plural detecting means may be optionally selected. The spectrophotometer for measuring absorbancy of light of plural wavelengths can be utilized in accordance with this invention. As such, a single apparatus is able to give plural detection signals representing spectral absorbancy and such absorbance signals provide very useful information about the identity of sample components.
As stated above, when plural detecting means are employed as detectors for a chromatograph, analytical information obtained depends upon differences in the sensitivity of the detector components in addition to the holding time with respect to separated sample components. It is exceedingly advantageous to distinguish component materials or peaks having components separated insufficiently.
The employment of plural detectors for use in a chromatograph has a problem, however, in that subsequent analytical processing of data is troublesome because there is such a large quantity of data. An object of this invention is to resolve this difficulty. In addition, plural detecting means are optionally selected. For instance, when a spectrophotomeric method is applied to predetermined light of plural wavelengths, detection data can be obtained for each selected wavelength. Thereby similar results as obtained in cases of employing plural detecting means can be obtained despite employing only one spectrophotometer.
For instance, when spectrophotometric analysis is applied continuously to chromatograph column effluent, records of change in absorption coefficient are obtained for light in several selected wavelengths. In these records, the set of absorbancy values for light in each wavelength at a given time can be regarded as one vector. This vector is recorded and its variation with time is investigated. Thereby provision of information from the recorded data is made much easier rather than by recording a change in many absorbancies. When considering a chromatogram as one traced pattern, its pattern recognition can be made easily.
Now, one peak of a chromatogram is taken into consideration, and this peak is assumed to be one wherein a component is completely separated. Since its separation is complete, this peak represents a change in concentration of a single component, and the value of absorbancy for light in predetermined plural wavelengths varies with the change in concentration from the peak rise to its fall; but a ratio of absorbancy among said wavelengths remains unchanged.
That is to say, a vector comprising vector components having absorbancy for each wavelength extends in its absolute value gradually from zero to an extreme maximum and thereafter becomes zero again, but the vector direction does not vary, and vector traces of change with time are expressed by a straight line in a direction, whose length alone is extended or contracted with time. The straight line vector represents a single sample component fully eluted.
Conversely, when vector tips of absorbancy depict a curved locus with time, it can be judged that a shown peak does not represent a single component. When taking two wavelengths, the vector is two-dimensional, and its change with time can be recorded by means of an X-Y recorder. FIG. 7 shows records of vector changes in this case, and FIG. 8 shows an ordinary chromatogram, whose three peaks I, II and III correspond to vector records I, II and III respectively in FIG. 7. The peaks I and III are the ones having one component completely separated, and their vector records are of one straight line as shown by I and III respectively in FIG. 7. Since the components representing these respective peaks are dissimilar, the ratios of absorbancy for two wavelengths differs. Accordingly I and III in FIG. 7 are shown with straight lines in different directions.
The peak II is one where components are not separated completely, and its vector record becomes a curved line as shown by II in FIG. 7. This peak comprises two components, and its vector components are considered to be tangents a and b at the origin of said curve. Although the peaks of these components are superimposed, the times when these peaks appear are offset from each other, and at the peak rise and the peak fall a single component exists. During development of the peak, two components exist in a mixed state while changing the ratio of concentration. Therefore, its vector record becomes a curve, for instance, that shifts gradually from vector a to vector b.
When one peak is formed by superposition of three component peaks, its vector record has three protrusions as shown in FIG. 9. In this case, components a, b and c constitute this peak, and this figure shows that elution is performed in a sequence of a, b, and c, or c, b, and a.
When detecting means of a number n where n&gt;2 are used, the vector record has to be made in such a manner that only two data are taken from n data and are recorded on a X-Y recorder as two-dimensional, and a maximum number (n-1) of two-data sets are processed for recording.